Although cytochrome c has been studied extensively since its rediscovery by Kelin in the 1920s, many uncertainties remain regarding the manner in which its functional properties are determined by its structure. These questions remain largely from the inability of chemical modification techniques to probe many types of amino acid residues and from the structural ambiguities that invariably occur in comparative studies of proteins from different species. Recent advances in molecular genetics now provide a new method of addressing these concerns through the application of oligodexyribonucleotide-directed site-specific mutagenesis. This approach, in principle, permits the replacement of any residue in any position of the protein sequence with any other naturally occurring amino acid residue if a functional clone of the gene coding for the protein is available. Previous work in our laboratories has employed or, in fact, developed all of the genetic and physical techniques relevant to this proposal. This experience will now be directed to preparing and characterizing the functional properties of five types of mutant yeast iso-l-cytochromes c which we have classified on the basis of the functional features of the protein that they are designed to probe. These classes of mutants include axial ligand mutants, heme thioether linkage mutans, hydrogen-bonding mutants, redox partner recognition mutants, and high-pH transition mutants. Initial studies in our laboratories have demonstrated that these techniques can readily generate 15-30 mg of mutant protein from 20 L cultures of yeast and have demonstrated that mutant cytochromes produced by these techniques provide a powerful means for studying the effect of cytochrome structure on the function of the protein. Combined, the initial results reported unambiguously demonstrate the feasibility and potential productivity of the work proposed.